We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submicromolar levels after 48–72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G2-M phase of the cell cycle after compound treatment. However, an increased sub-G1 population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel histone deacetylase inhibitor is currently in clinical trials as an anticancer agent.

Coordinated and precise regulation of gene expression is critical for maintenance of normal cell growth and differentiation (reviewed in Ref. 1). In eukaryotic cells, DNA is packaged into chromatin, and to alter gene expression, a dynamic process is required: local remodeling of nucleosomes (1). Reversible acetylation is one such process facilitated by histone acetyltransferases and histone deacetylases (HDACs). Transcriptionally active chromatin regions, such as the euchromatin, are associated with hyperacetylated histones, whereas transcriptionally silent heterochromatin regions are generally hypoacetylated (reviewed in Ref. 1). Both histone acetyltransferases and HDACs are recruited to target genes in complexes with sequence-specific factors and cofactors to regulate gene expression and ultimately cell function.

Inappropriate expression of genes required for cell proliferation, differentiation, or tumor suppression has been linked to cancer. Several lines of evidence suggest that aberrant recruitment of HDACs and the resulting chromatin modifications may lead to changes in gene expression seen in transformed cells: (a) silencing of tumor suppressor genes at the chromatin level (2, 3, 4, 5, 6, 7, 8); (b) interaction of HDAC-containing complexes with proteins involved in tumorigenesis (9, 10, 11); (c) reports of HDAC inhibitors (HDACIs) having significant antiproliferative effects, such as promoting differentiation, cell cycle arrest, or apoptosis (12, 13, 14, 15, 16, 17); and (d) induction of key mediators of G1 cell cycle arrest and differentiation, such as p21 with HDACIs (12, 18, 19, 20, 21). Studies of hematological malignancies also support involvement of HDACs in cancer development and/or maintenance. Aberrant recruitment of HDAC complexes is necessary for the carcinogenic properties of the fusion proteins PLZF-RARα, PML-RARα, and AML1/ETO in acute leukemia (reviewed in Ref. 22). In t(8;21) acute myelocytic leukemia (AML), HDAC-mediated repression of target genes such as AML1blocked differentiation of hematopoietic precursors (23, 24). In acute promyelocytic leukemia with t(11;17)/PLZF-RARαtranslocation, a combination of all-trans retinoic acid with the HDACI trichostatin resulted in differentiation of all-trans retinoic acid-resistant acute promyelocytic leukemia cells (25). Furthermore, treatment of leukemic blasts obtained from non-acute promyelocytic leukemia AML patients with a combination of retinoic acid and trichostatin induced terminal myeloid differentiation (26).

Preclinical experiments using small molecule inhibitors of HDACs, such as MS-275 and suberoylanilide hydroxamic acid (SAHA), exhibited efficacy against several human tumor xenografts in athymic mice (19, 27). In addition, the natural product HDACIs of the trapoxin class and trichostatin were shown to activate the p21 promoter, increase p21 protein levels, inhibit cyclin-dependent kinase 2 kinase activity, reduce retinoblastoma (Rb) phosphorylation, and cause cell cycle arrest or apoptosis in three human tumor cell lines (21, 28, 29). NVP-LAQ824, our novel synthetic HDACI, showed similar properties at submicromolar concentrations. Our studies also indicated that NVP-LAQ824 induced apoptosis in tumor cells but cell cycle arrest in normal fibroblasts. Moreover, increased histone acetylation in NVP-LAQ824-treated cancer cells confirmed the activity of our compound against HDACs. We therefore conclude that the antitumor effect seen was, in fact, attributable to specific inhibition of HDAC.

Materials.

NVP-LAQ824 was prepared in-house as the lactate salt and dissolved in DMSO. MS-275 was prepared as described in Suzuki et al.(29) as a free base and was dissolved in DMSO. Suberoylanilide hydroxamic acid (SAHA) was prepared as described in Richon et al.(30) and was dissolved in DMSO. The ECF Western blotting reagent pack for mouse or rabbit was purchased from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). Pre-cast NuPAGE gels were from Invitrogen Life Technologies, Inc. (Carlsbad, CA). The CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay was from Promega (Madison, WI). The ApoAlert Annexin V Apoptosis kit was from Clontech Laboratories, Inc. (Palo Alto, CA). The Hybond-P, polyvinylidene difluoride, membrane was purchased from Amersham Biosciences (Piscataway, NJ).

Cell Culture.

H1299, HCT116, A549, DU145, PC3, and MDA435 cells were obtained from American Type Culture Collection (Rockville, MD) and were maintained according to the supplier’s instructions. Normal dermal human fibroblast (NDHF) and normal human bronchial epithelial (NHBE) cells were obtained from Clonetics (San Diego, CA) and were maintained in DMEM supplemented with 15% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and BEGM BulletKit (Clonetics, San Diego, CA) supplemented with retinoic acid. hTERT/SV40 T antigen-transformed NHBE (hTERT/SV40 NHBE) cells were a kind gift from Dr. Barrett Rollins (Dana-Farber Cancer Institute, Harvard Medical Center, Boston, MA) and were maintained in BEGF BulletKit supplemented with retinoic acid.

Enzyme Purification and in Vitro Histone Deacetylase Assay.

HDAC enzymes were partially purified from H1299 cell lysate by ion exchange chromatography using the Q Sepharose Fast Flow column (Amersham Biosciences). The assay was performed as described previously (20) using the partially purified cell lysate and 3H-labeled acetylated histone-H4 peptide as the substrate. All enzymatic assays were performed in triplicate and indicated as the mean ± SD.

p21 Promoter Activation Assay.

The plasmid encoding the luciferase gene driven by the p21 promoter and its transfection into H1299 cells were described previously (20). Data were analyzed by calculating mean fold activation (n = 3) of compound-treated cells compared with the average of DMSO-treated control (n = 3). The percentage of activity for each concentration of NVP-LAQ824 was determined by comparison with the maximal fold activation obtained from psammaplin A, a natural product identified from a screen for p21 promoter activation (32).1 The AC50s were calculated using a linear regression calculation between data points to predict the concentration of compound needed for 50% activation relative to the reference.

Monolayer Growth Inhibition Assay.

Cell proliferation was measured using an adaptation of published procedures (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assay) as described previously (29). The CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay was performed according to the manufacturer’s protocol. The average background value (treatment with medium alone) was subtracted from each experimental well; triplicate values were averaged for each compound dilution. The following formulas were used to calculate the percentage of growth:

\[If\ X\ {>}\ T_{0},\ \%\ \mathrm{Growth}\ {=}\ ((X\ {-}\ T_{0})/(GC\ {-}\ T_{0}))\ {\times}\ 100\]
\[If\ X\ {<}\ T_{0},\ \%\ \mathrm{Growth}\ {=}\ (X\ {-}\ T_{0})/T_{0}\ {\times}\ 100\]

where T0 is the average value of T0 − background, GC is the average value of untreated cells (in triplicate) − background, and X is the average value of compound-treated cells (in triplicate) − background.

The “% Growth” was plotted against compound concentration and used to calculate the IC50 using the linear regression techniques between data points to predict the concentration of compounds at 50% inhibition.

Cell Cycle Analysis.

HCT116 and NDHF cells (1 × 106 each) were seeded on 10-cm2 tissue culture dishes and were allowed to grow overnight at 37°C with 5% CO2. HCT116 and NDHF cells were exposed to 0.1 and 0.5 μm NVP-LAQ824, respectively, or 0.1% DMSO. In addition, HCT116 cells were treated with 1 μm SAHA and 10 μm MS-275. After 24 and 48 h, the cells were collected, briefly resuspended in ice-cold 70% ethanol, and incubated in propidium iodide solution (70 μm propidium iodide, 38 mm sodium citrate, and 20 μg/ml RNase A) at 37°C for 30 min. Flow cytometric analysis was performed on a FACSort instrument (Becton Dickinson Immunocytometry Systems, San Jose, CA), and the data were subsequently analyzed using the ModFitLT software (Becton Dickinson).

Apoptosis Detection.

A549, HCT116, and NDHF cells (3 × 105 each) as well as NHBE and human telomerase reverse transcriptase (hTERT)/SV40 NHBE cells (1.5 × 105 each) were seeded in a 24-well plate (Costar Inc., Corning, NY). Twenty-four h later, cells were incubated with NVP-LAQ824 at the indicated concentrations or 0.1% DMSO as the control. Twenty-four and/or 48 h after NVP-LAQ824 treatment, the medium was removed, and the cells were incubated in the dark for 15 min with binding buffer containing 1 μg/ml Annexin V-FITC and 2.5 μg/ml propidium iodide. The cells were observed with a fluorescent microscope using a dual filter set for FITC and rhodamine.

Western Blot Analysis.

HCT116 and A549 tumor cells (1 × 106 each) were treated with the indicated concentrations of NVP-LAQ824 for the appropriate times. The treated cells were lysed in 500 μl of ice-cold triple detergent buffer [50 mm Tris-Cl (pH 8.0), 150 mm NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate, and 10 μl/ml protease inhibitor mixture). Cell lysates (10 μg) were separated on a 4–12% NuPAGE gel by electrophoresis and transferred onto the Hybond-P, polyvinylidene difluoride, membrane. The membranes were probed with anti-p21waf1 antibody (Oncogene Research Products, San Diego, CA) diluted 1:100, anti-Rb antibody (PharMingen, San Diego, CA) diluted 1:1000, anti-acetylated H3 antibody (Upstate Technologies, Lake Placid, NY) diluted 1:1000, anti-acetylated H4 antibody (Upstate Technologies, Lake Placid, NY) diluted 1:250, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Chemicon Inernational Inc., Temecula, CA) diluted 1:1000 for 2 h at room temperature or overnight at 4°C. The specific protein signals were detected using the ECF Western blotting reagent pack and the Storm 860 Phosphorimager (Molecular Dynamics, Sunnyvale, CA) after appropriate secondary antibody incubation.

Tumor Xenografts Implantation.

The studies were performed on-site, using outbred athymic (nu/nu) female mice (Hsd:Athymic Nude-nu from Harlan Sprague Dawley, Indianapolis, IN). Mice were anesthetized with Metofane (Mallinckrodt Veterinary, Inc., Mundelein, IL), and a cell suspension (100 μl) containing 1 × 106 HCT116 cells was injected s.c. into the right axillary (lateral) region of each animal. Tumors were allowed to reach the volume of approximately 100–400 mm3. At this point, mice bearing tumors with acceptable morphology (non-necrotic) and of similar size range were selected and distributed into groups of six for the studies. NVP-LAQ824 was dissolved in DMSO to create a stock solution, which was further diluted just before dosing with D5W to a final DMSO concentration of 10%. Tumor-bearing mice were treated with the compound by i.v. injection into the tail vein. NVP-LAQ824 was dosed once daily, 5 days/week, for a total of 15 doses. 5-Fluorouracil was administered at 100 mg/kg in 0.9% saline 1 day/week for a total of three doses. The control groups were treated with the vehicle. Tumors were collected from the animals at the indicated time points.

Statistical Analysis.

All in vitro experiments were conducted in triplicate and repeated at least three times. The results are presented as mean activity values, IC50 or AC50, ± SD of the mean. Animal experiments were analyzed using the Student’s t test for independence and were performed using the Sigmaplot software. P < 0.05 was determined to be significant.

NVP-LAQ824 Is a Potent HDACI in Vitro.

We have designed and synthesized a novel HDACI, which we termed NVP-LAQ824 (33). NVP-LAQ824 is a cinnamic acid (structure shown in Fig. 1) that belongs to the hydroxamic acid-based group of HDACIs that includes trichostatin A, SAHA, and pyroxamide. Currently, several of these inhibitors are in clinical trials as cancer therapeutics (34, 35, 36, 37, 38).

The effect of NVP-LAQ824 on human HDAC activity was determined by in vitro HDAC enzyme assays. The 50% inhibitory concentration for NVP-LAQ824 was remarkably similar over several independent experiments and was determined to be at a low nanomolar concentration (Table 1). Thus, NVP-LAQ824 is a potent inhibitor of HDAC activity.

NVP-LAQ824 Activated the p21 Promoter and Inhibited Cell Proliferation.

Inhibitors of HDACs activate the expression of the gene encoding the p21 cell cycle inhibitor (18, 19, 20, 39). To determine the effect of NVP-LAQ824 on p21 gene expression, transcriptional activation of a transiently transfected p21 promoter driving a luciferase reporter gene was measured in H1299 cells. The concentration of NVP-LAQ824 producing 50% of the maximal promoter activation (AC50) relative to psammaplin A, a natural product identified in a screen for inducing the p21 promoter, was shown in Table 1. These results demonstrated that nanomolar concentrations of NVP-LAQ824 were needed to induce the reporter construct, indicating specificity of the compound for the p21 promoter.

Previous studies have demonstrated that HDACIs induce tumor cell growth arrest; therefore, we examined the effects of NVP-LAQ824 on the growth of tumor cell lines. Monolayer growth inhibition assays were performed (Tables 1 and 2). Results of these experiments revealed that continuous exposure to NVP-LAQ824 for 72 h inhibited the growth of both H1299, a non-small cell lung carcinoma line, and HCT116, a colon cancer cell line. The calculated IC50s were 0.15 and 0.01 μm in H1299 and HCT116 cells, respectively. In contrast, the IC50s for another HDACI, MS-275, were much higher. In addition, we examined the effects of NVP-LAQ824 in two prostate cancer cell lines (DU145 and PC3) and a breast cancer line (MDA435). The IC50s in those cells were also in the nanomolar range (Table 2). Thus, low nanomolar concentrations of NVP-LAQ824 were sufficient to significantly inhibit growth of many tumor cells.

The Antiproliferative Effect of NVP-LAQ824 Is Selective to Human Tumor Cells.

For the potential use of NVP-LAQ824 as an anticancer agent, it must exhibit specificity for tumor cells at therapeutic doses (19). To determine whether NVP-LAQ824 is selective for cancer cells, NDHFs, colon carcinoma (HCT116), and non-small cell lung carcinoma (A549) cells were exposed to various concentrations of NVP-LAQ824, and cell viability was measured at different time points (Table 3). In addition, antiproliferative effects of NVP-LAQ824 were measured with respect to duration of treatment. All three cell lines were exposed to NVP-LAQ824 for 3, 6, 16, 24, 48, and 72 h before the compound was washed out, and net cell growth was measured after 72 h (Fig. 2). For both experiments, LD50 and LD90 values were measured and defined as the concentrations of compound that caused 50 and 90% cell death, respectively. The results of these experiments showed that continuous exposure of NVP-LAQ824 for 72 h produced LD90s of 0.09 μm in HCT116 cells and 0.47 μm in A549 cells (Table 3). Moreover, continuous treatment for up to 16 h, followed by incubation in drug-free medium, resulted in reduced net cell growth but not cell death. However, lethal effects were observed in HCT116 (Fig. 2,A) and A549 (Fig. 2,B) cells after 24 h of compound treatment. Interestingly, the LD90 was not achieved in either experiment with NDHFs, even at micromolar concentrations of NVP-LAQ824 tested (Table 3; Fig. 2,C). Generally, HCT116 cells were more sensitive than A549 cells, with NDHFs having the least sensitivity to the growth-inhibitory effects of NVP-LAQ824 (Fig. 2 D). These results indicated that the lethal effect of NVP-LAQ824 was time-dependent and exhibited a selective toxicity toward the tumor cell lines while inducing only growth arrest in normal fibroblasts.

Lack of G1-S Arrest in Tumor Cells Despite Induction of p21 Expression and Rb Hypophosphorylation by NVP-LAQ824.

Increased expression of p21 results in inhibition of cyclin-dependent kinase activity and consequent hypophosphorylation of cyclin/cyclin-dependent kinase substrates such as Rb. Hypophosphorylation of Rb is associated with cell cycle arrest at G1-S phase (12, 20). Consistent with previous findings, we observed a dose-dependent increase of p21 protein in A549 cells in response to NVP-LAQ824 treatment. Concomitant to the increased p21 level, we detected an increase in the hypophosphorylated state of the Rb tumor suppressor (Fig. 3). Thus, similar to many other HDACIs, NVP-LAQ824 was able to alter the expression level and change posttranslational modifications of key cell cycle regulators.

In light of the above findings as well as the observation that NVP-LAQ824 produced growth inhibition in cancer cell lines, we compared the cell cycle profiles of a normal cell line NDHF and a tumor cell line HCT116 subsequent to compound treatments. DMSO-treated cells were used as the negative control. NVP-LAQ824 treatment of NDHF cells caused the expected G1-S growth arrest in addition to a significant reduction of cells in S-phase and accumulation of cells at the G2-M checkpoint. These observations persisted through the 48-h treatment period without significant increases in sub-G1 population (Fig. 4,A). Exposure of HCT116 cells to HDACI revealed a surprising result. Treatment of HCT116 cells for 24 h with two other HDACIs, SAHA and MS-275, exhibited results consistent with the NDHF cells: G1-S arrest, decreased S-phase, and G2-M block. However, 24-h exposure of HCT116 cells to NVP-LAQ824 displayed no arrest at the G1-S boundary despite experimental observations of p21 induction and Rb hypophosphorylation in tumor cells (Fig. 4,B). These cells, nonetheless, maintained a significant decrease of S-phase and G2-M cell cycle arrest. Moreover, longer treatment of HCT116 cells with all three inhibitors triggered loss of G2-M arrest accompanied by moderate to significant increased sub-G1 populations (depicted by the blue-shaded areas), and this result was most profound with NVP-LAQ824 treatment (Fig. 4 B). Furthermore, the concentration of SAHA and MS-275 required to achieve these effects were 10-fold or more than levels of NVP-LAQ824. Similar results were observed with a non-small cell lung carcinoma cell line, A549 (data not shown). These results suggested that NVP-LAQ824 may have a slightly different mechanism of action and may be more potent than the other two inhibitors. The results also suggest NVP-LAQ824 treatment breached/bypassed Rb-associated checkpoint mechanisms normally responsible for the G1-S arrest, and all three treatments eventually breached G2-M checkpoints in the tumor cells. Contrary to tumor cells, these checkpoint controls appear likely to remain intact in treated normal fibroblasts.

Selective Induction of Apoptosis by NVP-LAQ824 in Human Cancer Cells.

The increased sub-G1 population observed by flow cytometry in tumor cells suggested that these cells might be undergoing apoptosis. To test this hypothesis, cells were stained with Annexin V, a marker for early stages of apoptosis (green fluorescence) and propidium iodide (red fluorescence) to detect apoptotic cell death. Our data showed that a significant percentage of A549 cells (Fig. 5,A) and the majority of HCT116 cells (Fig. 5,B) stained positive with Annexin V after 24 h of NVP-LAQ824 exposure. By 48 h, the majority of A549 and HCT116 cells stained positive with Annexin V and/or propidium iodide, indicating that NVP-LAQ824 induced apoptotic death in these tumor cells (Fig. 5, A and B). In contrast, NDHF cells did not show noticeable Annexin V staining at the 24-h time point, and limited staining was observed after 48 h of treatment (Fig. 5,C). A similar difference in apoptotic sensitivity to NVP-LAQ824 was observed between hTERT/SV40 T antigen transformed and untransformed human bronchial epithelial cells where Annexin V-stained cells were detected only in transformed cells (Fig. 5 D). Thus, unlike cancer cells, normal fibroblasts most likely underwent nonlethal growth arrest upon NVP-LAQ824 treatment, as suggested by the results of cell cycle profiling and experiments staining for a marker of apoptosis. Taken together, our data indicated that NVP-LAQ824 selectively induced apoptosis in tumor cells while arresting normal cells at the G1-S and G2-M checkpoints.

NPV-LAQ824 Increased Acetylation of Histones H3 and H4.

To confirm that the effects of NVP-LAQ824 observed in cultured cells were attributable to inhibition of HDACs, we examined the acetylation state of histones in HCT116 and A549 cells after treatment with NVP-LAQ824 (Fig. 6). Western blotting analyses using antibodies specific for acetylated histone H3 and H4 revealed a dramatic increase in the amount of histone acetylation after NVP-LAQ824 exposure for 3 h. Furthermore, the increase in acetylation reached a plateau at ∼0.02 μm NVP-LAQ824. In contrast, the amount of GAPDH was not affected under the same experimental conditions (Fig. 6). These results suggested that NVP-LAQ824 acted as a potent HDACI in human cancer cell lines.

NVP-LAQ824 Exhibited Antitumor Activities in HCT116 Tumor Xenografts in Athymic Nude Mice.

To assess the ability of NVP-LAQ824 to inhibit tumor growth, we examined its effects in s.c. implanted tumors in athymic nude mice. Tumors arose from HCT116 cells and reached an average size of 100 mm3 before NVP-LAQ824 or vehicle was administered i.v. once daily, 5 days/week for a total of 15 doses. Tumor sizes were measured every 7 days after implantation. As shown in Fig. 7, NVP-LAQ824 treatment produced a dose-dependent inhibition of tumor growth; at 100 mg/kg, its antitumor effect was similar to that of 5-Fluorouracil, a standard anticancer agent. Importantly, no significant changes in animal body weight were observed even at the highest compound concentration, suggesting that the growth-inhibitory effect was tumor specific and that NVP-LAQ824 did not produce a general cytotoxicity in mice.

In this report, we describe the effects of a novel HDACI, NVP-LAQ824, in cultured tumor cells. NVP-LAQ824 inhibited the HDAC enzyme purified from tumor cells and activated the p21 promoter at low nanomolar concentrations. The compound also exhibited highly potent antiproliferative effects, including apoptosis in transformed cells and cell cycle arrest in normal fibroblasts.

NVP-LAQ824 treatment of tumor cell lines HCT116 and A549 and normal fibroblasts (NDHFs) generated different growth and apoptotic profiles. In HCT116, a tumor cell line, low nanomolar concentrations of NVP-LAQ824 initially induced only G2-M arrest, but continued exposure for more than 24 h triggered progression to apoptosis. Incubation of HCT116 cells with other known inhibitors of HDACs, SAHA and MS-275, led to both G1-S and G2-M arrest followed by apoptosis. In contrast to NVP-LAQ824, the growth inhibition and apoptosis of HCT116 cells induced by SAHA and MS-275 were observable only when the cells were exposed to levels that were 10-fold or greater than concentrations of NVP-LAQ824 that promoted cell death. Upon several-fold higher NVP-LAQ824 exposure, NDHFs underwent a complete loss of S-phase and accumulated at the G1-S and G2-M boundaries, with minimal cell death. Moreover, the apoptotic study comparing the effects of NVP-LAQ824 on two normal cell types (fibroblast and epithelial) and two transformed epithelial cells support a sensitivity window between normal and malignant cells during NVP-LAQ824 treatment. After 24-h treatment with NVP-LAQ824, the transformed cells stained for a marker for apoptosis, while the normal cells remained unstained. It is interesting to note that despite p21 induction and hypophosphorylation of Rb, NVP-LAQ824 did not produce significant G1-S arrests in transformed cells compared with G1-S-arrested normal cells. We speculate that eventual death of tumor cells may be attributed to their inability to stably arrest. Preliminary studies from our laboratory have shown that differential cellular sensitivity toward NVP-LAQ824 and other HDACIs were associated with differential expression of mitotic checkpoint regulators (data not shown). Other reports have also implicated malfunction of G2-M checkpoint mediator(s) in transformed cell lines as the culprit for the loss of growth arrest after HDACI treatment (40). Combining this study with other reports, a model can be drawn to explain behaviors of tumor cells versus normal cells to NVP-LAQ824 exposure, and the key may lie with identifying the defect(s) in both G1-S and G2-M checkpoint mechanisms. If these observations seen in NDHF cells represent response of other normal cell types to NVP-LAQ824, there might be a favorable therapeutic index between normal and tumor cells in vivo. Our studies in mice have shown that NVP-LAQ824 was able to inhibit tumor growth without producing general cytotoxicities in these animals.

The induction of apoptosis by NVP-LAQ824 appears to be p53 independent because cell death occurred in MDA435 (p53 mutant), HCT116, and A549 cells (both p53 wild type) but not in NDHFs (p53 wild type) cells. In addition, previous reports have shown that activation of p21 gene expression by HDACIs was independent of the p53-binding element at the p21 promoter (20). The mechanism by which NVP-LAQ824 triggers apoptosis in tumor cells is currently unknown; however, studies have shown increased expression of CD95/Fas or tumor necrosis factor-related apoptosis-inducing ligand, key components of the apoptotic pathways, in response to HDACI (41, 42).

The growth-inhibitory effects of NVP-LAQ824 were time dependent and required at least 16 h to produce lethality in tumor cells. Removal of the compound in <16 h rescued cells from death. These findings suggest that NVP-LAQ824-induced cell killing is a controlled specific event that probably requires changes in gene expression before an irreversible trigger forces tumor cells to their fates. This hypothesis is consistent with the mechanism of action for inhibitors of HDACs that are likely to mediate their effects through alterations in the transcriptional regulatory processes. Furthermore, the ability to detect changes in the acetylation state of histones may serve as a biomarker to examine clinical samples. In conclusion, the novel HDACI, NVP-LAQ824, offers the potential to be a highly potent anticancer therapeutic.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Peter Atadja, Oncology Molecular and Cellular Biology Unit, Novartis Institutes for Biomedical Research, East Hanover, NJ 07936. Phone: (862) 778-0435; Fax: (973) 781-7578; E-mail: peter.atadja@pharma.novartis.com

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Perez, L. B., Atadja, P., Bair, K., Cornell, W., Dean, K., Green, M., Kwon, P., Lamberson, C., Remisewski, S., Sambucetti, L., and Trogani, N. Exploring the structure-activity relationships of psammaplins: a new class of histone deacetylase inhibitors, manuscript in preparation.

Fig. 1.

Chemical structure of NVP-LAQ824.

Fig. 1.

Chemical structure of NVP-LAQ824.

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Fig. 2.

Time-dependent effects of NVP-LAQ824 on cell growth and viability. The experiments were performed as described in Table 3. All experiments were conducted in triplicate and repeated at least three times. Data points are percent mean growth ± SD. A, treatment in HCT116 cells. B, treatment in A549 cells. C, treatment in NDHF cells. D, compilation of all three cell lines from 48-h exposure to NVP-LAQ824.

Fig. 2.

Time-dependent effects of NVP-LAQ824 on cell growth and viability. The experiments were performed as described in Table 3. All experiments were conducted in triplicate and repeated at least three times. Data points are percent mean growth ± SD. A, treatment in HCT116 cells. B, treatment in A549 cells. C, treatment in NDHF cells. D, compilation of all three cell lines from 48-h exposure to NVP-LAQ824.

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Fig. 3.

NVP-LAQ824 induces p21 protein expression and Rb hypophosphorylation. A549 cells were treated with NVP-LAQ824 at the indicated concentration for 48 h. Total cell lysates were analyzed by Western blotting using antibodies for p21 (upper panel) and Rb (lower panel).

Fig. 3.

NVP-LAQ824 induces p21 protein expression and Rb hypophosphorylation. A549 cells were treated with NVP-LAQ824 at the indicated concentration for 48 h. Total cell lysates were analyzed by Western blotting using antibodies for p21 (upper panel) and Rb (lower panel).

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Fig. 4.

Cell cycle alterations in response to NVP-LAQ824 treatment. NDHF cells were treated with NVP-LAQ824 (0.5 μm) for 24 and 48 h. In addition, HCT116 cells were treated with NVP-LAQ824 (0.1 μm), MS-275 (10 μm), and SAHA (1 μm) for 24 and 48 h. Cell cycle profiles were analyzed by flow cytometry, and the percentage of the cells in G0-G1, S, and G2 phases were calculated by the ModFit program and are shown. A, cell cycle profiles of NDHF cells at 24 and 48 h. B, cell cycle profiles of HCT116 cells at 24 and 48 h.

Fig. 4.

Cell cycle alterations in response to NVP-LAQ824 treatment. NDHF cells were treated with NVP-LAQ824 (0.5 μm) for 24 and 48 h. In addition, HCT116 cells were treated with NVP-LAQ824 (0.1 μm), MS-275 (10 μm), and SAHA (1 μm) for 24 and 48 h. Cell cycle profiles were analyzed by flow cytometry, and the percentage of the cells in G0-G1, S, and G2 phases were calculated by the ModFit program and are shown. A, cell cycle profiles of NDHF cells at 24 and 48 h. B, cell cycle profiles of HCT116 cells at 24 and 48 h.

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Fig. 5.

Detection of apoptosis by Annexin V staining. A549 (0.5 μm), HCT116 (0.1 μm), and NDHF (0.5 μm) cells were treated with NVP-LAQ824 for 24 and 48 h. NHBE (1 μm) and hTERT/SV40 NHBE (0.1 μm) cells were treated with NVP-LAQ824 for 24 h. Cells were stained with Annexin V (green fluorescence) and propidium iodide (red fluorescence) as described in “Materials and Methods.” Experiments were repeated at least two times, and representative results are shown. A, treatment of A549 cells. B, treatment of HCT116 cells. C, treatment of NDHF cells. D, treatment of NHBE and hTERT/SV40 NHBE cells.

Fig. 5.

Detection of apoptosis by Annexin V staining. A549 (0.5 μm), HCT116 (0.1 μm), and NDHF (0.5 μm) cells were treated with NVP-LAQ824 for 24 and 48 h. NHBE (1 μm) and hTERT/SV40 NHBE (0.1 μm) cells were treated with NVP-LAQ824 for 24 h. Cells were stained with Annexin V (green fluorescence) and propidium iodide (red fluorescence) as described in “Materials and Methods.” Experiments were repeated at least two times, and representative results are shown. A, treatment of A549 cells. B, treatment of HCT116 cells. C, treatment of NDHF cells. D, treatment of NHBE and hTERT/SV40 NHBE cells.

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Fig. 6.

NVP-LAQ824 treatment increased histone H3 and H4 acetylation in tumor cells. HCT116 and A549 cells were treated with the indicated concentrations of NVP-LAQ824 for 3 h. Acetylation levels of histones H3 (Ac-H3) and H4 (Ac-H4) were analyzed by Western blotting. The amount of GAPDH was examined to ensure equal loading of samples in each lane.

Fig. 6.

NVP-LAQ824 treatment increased histone H3 and H4 acetylation in tumor cells. HCT116 and A549 cells were treated with the indicated concentrations of NVP-LAQ824 for 3 h. Acetylation levels of histones H3 (Ac-H3) and H4 (Ac-H4) were analyzed by Western blotting. The amount of GAPDH was examined to ensure equal loading of samples in each lane.

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Fig. 7.

Antitumor activity of NVP-LAQ824 in tumor xenografts in nude mice. Growth curves of HCT116 and human colon tumor xenografts are shown and expressed as mean tumor volume ± SE. The mice were treated with NVP-LAQ824 dissolved in the delivery vehicle (5% dextrose in water) or 5-Fluorouracil (in 10% DMSO/D5W) at the indicated concentrations. T/C represents the ratio of compound-treated tumor volume to vehicle-treated tumor volume, expressed as a percentage. The reduction of tumor volume between all compound-treated versus vehicle-treated animals showed statistical significance P < 0.05.

Fig. 7.

Antitumor activity of NVP-LAQ824 in tumor xenografts in nude mice. Growth curves of HCT116 and human colon tumor xenografts are shown and expressed as mean tumor volume ± SE. The mice were treated with NVP-LAQ824 dissolved in the delivery vehicle (5% dextrose in water) or 5-Fluorouracil (in 10% DMSO/D5W) at the indicated concentrations. T/C represents the ratio of compound-treated tumor volume to vehicle-treated tumor volume, expressed as a percentage. The reduction of tumor volume between all compound-treated versus vehicle-treated animals showed statistical significance P < 0.05.

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Table 1

In vitro activity profile of NVP-LAQ824

The in vitro enzyme inhibition, p21 promoter activation, and growth inhibition assays were performed with various concentrations of NVP-LAQ824 and MS-275. HDAC enzyme assays and IC50s from those assays were determined as described in “Materials and Methods.” The p21 promoter activation assays were done in triplicate, as described in “Materials and Methods,” and AC50 values are indicated. Inhibition of cell growth in monolayer after 72-h compound treatment was measured in triplicate by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethyl-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assays as described in “Materials and Methods.” All experiments were repeated at least three times, and the indicated values represent the mean of IC50 or AC50 ± SD.

CompoundMonolayer growth
HDAC enzymep21 promoter activationH1299HCT116
IC50m)SDAC50m)SDIC50m)SDIC50m)SD
NVP-LAQ824 0.032 0.018 0.30 0.20 0.15 0.015 0.010 0.002 
MS-275 >10 NAa 7.8 0.9 6.8 1.1 0.67 0.12 
CompoundMonolayer growth
HDAC enzymep21 promoter activationH1299HCT116
IC50m)SDAC50m)SDIC50m)SDIC50m)SD
NVP-LAQ824 0.032 0.018 0.30 0.20 0.15 0.015 0.010 0.002 
MS-275 >10 NAa 7.8 0.9 6.8 1.1 0.67 0.12 
a

NA, not applicable.

Table 2

Growth inhibitory effects of NVP-LAQ824 in several cancer cell lines

Tumor cells, as indicated, were grown in monolayers and treated with NVP-LAQ824 at various concentrations. IC50s were determined by 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethyl-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assays and performed in replicates of three. The numbers represent the mean IC50 s ± SD.

Cancer cell lines
H1299HCT116DU145PC3MDA435
NVP-LAQ824 IC50m0.15 0.010 0.018 0.023 0.039 
Cancer cell lines
H1299HCT116DU145PC3MDA435
NVP-LAQ824 IC50m0.15 0.010 0.018 0.023 0.039 
Table 3

Time-dependent effects of NVP-LAQ824 on cell viability

HCT116, A549, and NDHF cells were treated with various concentrations of NVP-LAQ824 for the indicated amount of time before the removal of the compound and the addition of fresh medium. Cell viability was measured at 72 h by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethyl-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assays as described in Table 1. Definitions of IC50, LD50, and LD90 were described in “Materials and Methods” and are indicated as the mean ± SD.

IC50m)LD50m)LD90m)
HCT116    
 24 h 0.01 0.64 2.26 
 48 h 0.01 0.04 0.14 
 72 h 0.01 0.04 0.09 
A549    
 24 h 0.05 >3.75 >3.75 
 48 h 0.03 0.20 0.95 
 72 h 0.03 0.16 0.47 
NDHF    
 24 h >3.75 >3.75 >3.75 
 48 h 0.13 >3.75 >3.75 
 72 h 0.04 0.61 >3.75 
IC50m)LD50m)LD90m)
HCT116    
 24 h 0.01 0.64 2.26 
 48 h 0.01 0.04 0.14 
 72 h 0.01 0.04 0.09 
A549    
 24 h 0.05 >3.75 >3.75 
 48 h 0.03 0.20 0.95 
 72 h 0.03 0.16 0.47 
NDHF    
 24 h >3.75 >3.75 >3.75 
 48 h 0.13 >3.75 >3.75 
 72 h 0.04 0.61 >3.75 
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